An intimate relationship exists between brain function and the brain vasculature. In the brain, the vasculature is engaged in active, bidirectional communication with neurons and astrocytes, collectively termed the neuro(glio)vascular unit. When local populations of neurons are active, coordinated activity in the neurovascular unit leads to increased blood flow and volume to the activated brain region, a process known as functional hyperemia. Endothelial cells are a specialized cell-type located within vessel walls that play a crucial role in regulation of the vasculature. Perhaps most prominent is their role as the primary substrate of the blood-brain barrier (BBB). Disrupting vascular endothelial cell (vEC) physiology can lead to breakdown of the BBB and development of several pathological conditions, including diabetes, multiple sclerosis, inflammatory pain, Alzheimer's disease, epilepsy, and stroke. Calcium (Ca2+) is generally recognized as a universal second messenger that contributes to essential cellular signaling events in a broad range of tissues and organisms. Depletion of extracellular Ca2+ levels or conditions that lead to abnormally high or low intracellular Ca2+ concentrations in vECs can lead to alterations in vascular responses and eventually to a breakdown in the BBB. Despite the clear importance of vEC Ca2+ signaling to vascular dynamics, to our knowledge there has never been a study of vEC Ca2+ dynamics in the brain in vivo, nor any direct examination of the natural processes or specific cell types that drive vEC dynamics. The aims of this proposal are to test the hypothesis that Ca2+ dynamics are spontaneously expressed in vECs, driven by natural (sensory) input, and driven by local and distal neuromodulatory activation in vivo. To this end, we optimized two new methods that will allow visualization of vEC Ca2+ dynamics using two- photon imaging of genetically-encoded Ca2+ indicators in neocortical vECs in awake animals. Preliminary data using this approach has led to the characterization of ongoing basal vEC Ca2+ oscillations in the brain. Further, for the first time, we have found that vEC Ca2+ dynamics can be evoked by physiological sensory input to the mouse barrel cortex. We propose to extend these preliminary findings and use cell-type specific optogenetic activation to functionally dissect the precise cellular components within local somatosensory and distal neural networks that contribute to sensory-evoked vEC Ca2+ dynamics in the brain. PUBLIC HEALTH RELEVANCE: The blood-brain barrier is a crucial component of the central nervous system that restricts the passage of potentially harmful substances from the bloodstream into the brain. Breakdown of the blood-brain barrier can lead to the development of several pathological conditions, including diabetes, multiple sclerosis, inflammatory pain, Alzheimer's disease, epilepsy, and stroke. Gaining a better understanding of the individual cells that comprise the blood-brain barrier has the potential to uncover new therapeutic approaches to treating these conditions and improving human health.